Technical Field
The present invention generally relates to microfluidic devices and methods for processing samples for molecular diagnostic applications, for example detection of target nucleic acid sequences.
Description of the Related Art
The role of molecular diagnostics is critical in today's global health care environment. In the developing world, 95% of deaths are due to a lack of proper diagnostics and the associated follow-on treatment of infectious diseases; i.e., acute respiratory infections (ARIs), malaria, HIV, and tuberculosis (TB) (Yager, P et al, Annu Rev Biomed Eng 10:107-144, 2008). Recent pandemics like the 2009 H1N1 Influenza A pandemic, have accentuated the need for tools to effectively detect and control infectious diseases. Factors like “rapid pathogen mutation rates, transformation of nonhuman pathogens into human pathogens, and recombination of non human pathogen with human pathogens” have added to the challenge of managing novel infectious diseases (Kiechle, F L et al., Clin Lab Med 29(3):555-560, 2009). Increased global mobility has aided the rapid spread of infectious diseases from region of origin to other parts of the world as seen during the 2009 H1N1 pandemic. This mobility has highlighted the need for rapid, portable diagnostic (point-of-care [POC]) devices at ports of entry to prevent global spread of infections. Current laboratory culture methods for pathogens take a day or more to provide results.
For certain other types of infections, in both the developed and developing worlds, the diagnostic tests need to be repeated periodically to measure response to therapy and monitor the disease condition. One such case is monitoring the viral load (number of viral particles per milliliter of blood) for infections like HIV (Human immunodeficiency virus) and hepatitis C. Sub-Saharan Africa is a region heavily affected by the AIDS pandemic. The lack of standard laboratory facilities and trained laboratory technicians in these regions is a serious bottleneck. Similar problems exist in medically underserved areas of the USA. Rapid, low-cost diagnostic tools that can be dispersed throughout a community for easy access, possibly even in the home, would provide substantial benefit by allowing more rapid diagnosis and monitoring of disease and infection.
Nucleic acid biomarkers are the target analytes for several infectious diseases of high global health importance, including HIV, HCV, HBV, pandemic influenza, and dengue. A major challenge in developing a simple diagnostic device to test multiple viral agents is that the genome of some viruses are comprised of DNA, while those of other viruses are comprised of RNA. A further challenge for RNA-based analytes is specimen handling that protects the integrity of these labile molecules. There are several commercially available products that address this latter problem. Most of these products are expensive, technically demanding, and/or require some form of refrigeration. These requirements cannot be easily met by miniaturized microfluidic devices with on-cartridge reagent reservoirs designed for rapid, on-site diagnostic analyses. Moreover, these requirements cannot be easily met in low-resource or remote settings, as is the case in the majority of the developing world. Thus, there is a need for a low-cost, non-instrumented, and simple-to-use diagnostic device that can be used to prepare stable samples of nucleic acids and analysis of both DNA and RNA biomarkers at the point of care (POC).
Blood is the human tissue routinely used for nucleic acid expression studies and blood-based biomarker analysis because it can be easily collected. However, whole blood often contains many factors, such as heme and heparin, which interfere with and/or inhibit, many downstream analytic procedures. Moreover, blood plasma is extremely high in ribonuclease (RNase) activity, and minimizing this activity is critical to any RNA isolation procedure. Although DNA can be prepared from clinical samples under harsh conditions and stabilized, for example, simply by spotting on filter paper and allowing to dry at room temperature, RNA preparation has typically required the use of stabilizing agents and refrigeration and/or freezing. The steps required to stabilize RNA in clinical samples are cumbersome and not amenable to microfluidic, “sample to answer” diagnostic devices.
Variations of two methods have historically been used to prepare RNA from biological samples: chemical extraction and immobilization on glass, often referred to as “solid-phase extraction.” Chemical extraction methods usually use highly concentrated chaotropic salts in conjunction with acidic phenol or phenol-chloroform solutions to inactivate RNases and purify RNA from other biomolecules. These methods provide very pure preparations of RNA; however, the RNA must typically be desalted and concentrated with an alcohol precipitation step. The solid-phase extraction method, described in U.S. Pat. No. 5,234,809 to Boom et al., relies on the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate together with the nucleic acid-binding properties of solid-state silica particles or diatoms in the presence of this agent. After silica-bound RNA is washed with a high-salt buffer containing ethanol, the RNA is eluted in a low-ionic-strength buffer.
It will be readily appreciated that sample preparation methods requiring aqueous extraction with organic solvents or chaotropic agents are tedious, hazardous, labor-intensive, and slow. Moreover, if great care is not taken in performing the procedures, residual contamination with nucleases can occur, and the sample nucleic acids will be degraded or lost. Diagnostic tests performed with such samples can give false negative results due to such degradation. False negative results can also be obtained due to chemical interference, for example from residual anionic detergents, chaotropic salts, or ethanol remaining in the sample and inhibiting target amplification procedures. If anionic detergents and proteases have been used, residual proteolytic activity can also degrade the enzymes used in target amplification and/or hybridization detection reactions and produce false negative results. Sample preparation methods based on the “Boom lysis” protocol disclosed in the '809 patent are commonly viewed as adequately addressing these problems. However, the present inventors have unexpectedly found that such extraction methods, utilizing chaotropic salts combined with solid-phase extraction, are not reliably effective in the preparation of blood or plasma samples for PCR-based detection of the HBV genome. Thus, none of the above-cited protocols is suitable for the preparation of a common sample for detection of both DNA and RNA targets from complex biological starting materials, e.g., whole blood and blood serum. This is particularly true for infectious disease diagnosis in clinical laboratory settings, where time demands are very high, and in low-resource areas where cost-effectiveness, reduction of toxic waste streams and simplicity are also of prime importance.
While progress has been made in the field, there continues to be a need in the art for point of care diagnostic devices, such as microfluidic devices, capable of isolating and analysis of nucleic acids from test samples. The present invention fulfills these needs and provides further related advantages.